Method for producing peptides and their salts which have an agonist activity of luteinizing hormone releasing hormones secreted from the hypothalamus

ABSTRACT

A peptide of the formula: 
     
       
         5-oxo—Pro—R 1 —Trp—Ser—R 2 —R 3 —R 4 —Arg—Pro—R 6   (I)  
       
     
     wherein R 1  represents His, Tyr, Trp or p—NH 2 —Phe, R 2  represents Tyr or Phe, R 3  represents an optionally substituted Gly or an optionally substituted α-D-amino acid residue, R 4  represents Leu, Ile or Nle, R 6  represents (1) Gly—NH—R 7 , wherein R 7  represents a hydrogen atom or an alkyl group which may optionally be substituted with a hydroxyl group, or (2) NH—R 8 , wherein R 8  represents a hydrogen atom, an alkyl group which may optionally be substituted with a hydroxyl group or an ureido group (—NH—CO—NH 2 ), is produced advantageously in an industrial scale by reacting a peptide of the formula: 
     
       
         5-oxo—Pro—R 1 —Trp—Ser—R 2 —R 3 —OH  (II)  
       
     
     wherein R 1 , R 2  and R 3  have the same meanings as defined above or its salts, with a peptide of the formula: 
     
       
         H—R 4 —R 5 —Pro—R 6   (III)  
       
     
     wherein R 4  and R 6  have the same meaning as defined above and R 5  represents Arg which has been protected or its salts, to produce a peptide of the formula: 
     
       
         5-oxo—Pro—R 1 —Trp—Ser—R 2 —R 3 —R 4 —R 5 —Pro—R 6   (I′)  
       
     
     wherein R 1 , R 2 , R 3 , R 4 , R 5  and R 6  have the same meanings as defined above or its salt, and then subjecting thus obtained peptide (I′) to a de-protecting group reaction.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to a method for producing peptides, which have anactivity of an agonist of luteinizing hormone releasing hormones (LHRH)secreted from the hypothalamus or their salts. The present inventionfurther relates to an intermediate peptide, its production method, itscrystals and a method for producing the crystals.

2. Description of the Related Art

U.S. Pat. No. 4,008,209 which corresponds to Japanese Patent ApplicationLaid-open No. 50-059370/1975, describes the following method forproducing a peptide of the formula: (Pyr)Glu—His—Trp—Ser—Tyr(orPhe)—X—Leu(or IIe, or Nle)—Arg—Pro—NH—R (SEQ ID NO: 13), wherein eachamino acid residue has L-configuration unless otherwise indicated; Xrepresents D—Leu, D—NLe, D—NVa, D—Ser, D—Abu, D—Phg, D—Phe or α—Aibu; Rrepresents an alkyl group which may optionally have hydroxyl.

In the reaction scheme, all symbols have the same meanings as definedabove.

Japanese Patent Application Laid-open No. 51-6926/1996, whichcorresponds to U.S. Pat. No. 3,997,516, describes a method for producinga peptide having a guanidino group which comprises protecting theguanidino group of a guanidino group-containing peptide with a loweralkoxybenzenesulfonyl group or tri(lower)alkyl-benzenesulfonyl group.

Furthermore, Japanese Patent Application Laid-open No. 51-100030/1996,which corresponds to U.S. Pat. No. 3,997,516, describes a method forproducing a peptide having a guanidino group which comprises protectingthe guanidino group of a guanidino group-containing peptide with a loweralkoxybenzenesulfonyl group or tri(lower)alkylbenzenesulfonyl group and,after the peptide condensation reaction, removing the protective groupwith a halogenosulfonic or lower alkylsulfonic acid or a Lewis acid.

For the production of a peptide on a commercial scale, it is essentialthat various parameters such as (1) qualities of starting materials, (2)production cost, (3) workability, (4) safety to operators, and (5)prevention of pollution should satisfy certain practically acceptablelevels and every process that is satisfactory at the laboratory level isnot necessarily satisfactory at the factory level. Therefore, design ofa commercial process for producing a peptide involves many problems thatmust be solved for satisfying a variety of requirements such as themodality of peptide chain extension; the selection of the site offragment condensation; the method for inhibiting isomerization atcondensation of fragments; the selection of protective groups for theα-position and side-chain functional groups; the method for finalelimination of such protective groups; the method for purification ofthe end product peptide; and the overall operability of the series ofsteps, among others.

Furthermore, a diversity of methods and a variety of reaction conditionsmay be contemplated for the synthesis of peptides in general but it isoften the case that because of non-crystallizability, many intermediatesused in the process cannot be sufficiently purified or requiretime-consuming fractionation procedures, with the result that manyprocesses are not satisfactory in the reproducibility of quality andyield. Thus, the physical characteristics such as crystallizability,stability, and solubility of intermediates, which are key factors in therespective production stages, hold sway over whether a process can becommercially acceptable in many cases.

Referring to the methodology for production of the peptide of theformula:(Pyr)Glu—His—Trp—Ser—Tyr(or Phe)—X—Leu(or Ile, orNle)—Arg—Pro—NH—R (SEQ ID NO: 13), the process disclosed in U.S. Pat.No. 4,008,209, which corresponds to Japanese Patent ApplicationLaid-open No. 50-059370/1975, protects the guanidino group of Arg withnitro and, therefore, the group Z used for protecting the α-amino groupcan hardly be reductively eliminated selectively, so that HBr—AcOH isused for removal of group Z. Inevitably, in this process, benzylbromide, which is highly lacrimetory, is by-produced in a large quantityand, moreover, a large amount of ether must be used for isolation of theend product. In addition, while the end product of this reaction isusually recovered in the hydrobromide form, the hydrogen bromide must beremoved with, for example, an ion exchange resin in order that theobjectionable isomerization on condensation of fragments may beinhibited. Furthermore, while the gradient elution method is used in thechromatographic purification of the final end product, the abovepeptide, the lower reproducibility of the concentration gradientnecessitates rigorous qualitative testing, with the result that theoperation for steady production of the desired product of uniformquality cannot be stand-ardized. Therefore, the process is not suitedfor industrial-scale production.

Furthermore, in the U.S. Pat. No. 3,997,516, a reaction scheme producesa peptide of the formula:(Pyr)Glu—His—Trp—Ser—Tyr—D—Leu—Leu—Arg(MBS)—Pro—NH—C₂H₅ (SEQ ID NO: 14)wherein MBS denotes p-methoxybenzenesulfonyl group. However, in the U.S.patent, only the reaction scheme is described, but other details such asreaction conditions are not described at all.

Thus, an industrially profitable process for producing peptides whichhave an activity of an aqonist of LHRH or its salts with safety,expedience, high yield and good reproducibility has not yet beendeveloped.

The inventors of the present invention explored the above-mentionedproblems with diligence and succeeded in establishing a protocol for theeffective production of intermediate peptide (II), mentioned below,through inhibition of isomerization of amino acid residues in thehydrolysis reaction step, a protocol for crystallization of the peptide(II); and deprotection on an industrial scale of peptide (I′), mentionedbelow; and a protocol for industrial-scale purification of the objectivepeptide (I), mentioned below. Accordingly, the inventors arrived at aprocess for producing said peptide (I) with safety, high yield, and goodreproducibility, and have completed the present invention.

SUMMARY OF THE INVENTION

The present invention is directed to

(1) A method for producing a peptide of the formula:

5—oxo—Pro—R¹—Trp—Ser—R²—R³—R⁴—Arg—Pro—R⁶  (I) (SEQ ID NO: 6)

wherein R¹ represents His, Tyr, Trp or p—NH₂—Phe, R² represents Tyr orPhe, R³ represents an optionally substituted Gly or an optionallysubstituted α-D-amino acid residue, R⁴ represents Leu, Ile or Nle, R⁶represents (1) Gly—NH—R , wherein R⁷ represents a hydrogen atom or analkyl group which may optionally be substituted with a hydroxyl group or(2) NH—R⁸, wherein R⁸ represents a hydrogen atom, an alkyl group whichmay optionally be substituted with a hydroxyl group or an ureido group(—NH—CO—NH₂), or its salts, which comprises reacting a peptide of theformula:

5—oxo—Pro—R¹—Trp—Ser—R²—R³—OH  (II) (SEQ ID NO: 4)

wherein R¹, R² and R³ have the same meanings as defined above or itssalts, with a peptide of the formula:

H—R⁴—R⁵—Pro—R⁶  (III) (SEQ ID NO: 15)

wherein R⁴ and R⁶ have the same meaning as defined above and R⁵represents Arg which has been protected, or its salts, to produce apeptide of the formula:

5—oxo—Pro—R¹—Trp—Ser—R²—R³—R⁴—R⁵—Pro—R⁶  (I′) (SEQ ID NO: 5)

wherein R¹, R², R³, R⁴, R⁵ and R⁶ have the same meanings as definedabove, or its salt, and then subjecting thus obtained peptide (I′) to ade-protecting group reaction,

(2) A method according to the item (1), wherein R¹ is His, R² is Tyr, R³is Gly, D—Leu, D—Trp, D—Val which may be substituted with C₁₋₄ alkyl,D—Ser, D—Ala which may be substituted with C₁₋₄ alkoxy, with naphthyl orwith 2-methylindolyl, or D—His which may be substituted with C₇₋₁₀aralkyl, R⁴ is Leu, R⁵ is Arg which is protected with a group selectedfrom the group consisting of a C₁₋₆ alkoxybenzenesulfonyl group,tri—C₁₋₆ alkylbenzenesulfonyl group and a nitro group, R⁶ is a group ofthe formula: NH—R⁸′, wherein R⁸′ is a hydrogen atom or an alkyl groupwhich may optionally be substituted with hydroxyl,

(3) A method according to the item (1), wherein R¹ is His, R² is Tyr, R³is D—Leu, R⁴ is Leu, R⁵ is Arg which is protected with a C₁₋₆alkoxybenzenesulfonyl group, R⁶ is a group of the formula: NH—R⁸″,wherein R⁸″ is a C₁₋₃ alkyl group which may optionally be substitutedwith hydroxyl,

(4) A method according to the item (1), wherein the reaction of thepeptide (II) or its salts with the peptide (III) or its salts is carriedout at a temperature ranging from about 0 to 40° C. for about 30 to 60hours,

(5) A method according to the item (1), wherein an acid is used in thede-protecting group reaction,

(6) A method according to the item (5), wherein the acid is C₁₋₆alkylsulfonic acid,

(7) A method according to the item (5), wherein the acid is used at aratio of about 5 to 25 times (weight) of the peptide (I′),

(8) A method for recovering and purifying the peptide (I) as defined inthe above item 1, which comprises subjecting the reaction mixturecontaining an emerged oily product of a free form to a purification ofcolumn chromatography, the reaction mixture being obtained by thereaction of de-protecting group reaction of the Peptide (I′) under theexistence of an acid and then neutralized with a base,

(9) A peptide of the formula:

5—oxo—Pro—R¹—Trp—Ser—R²—R³—OR  (IV) (SEQ ID NO: 3)

wherein R¹ is His, Tyr, Trp or p—NH₂—Phe, R² is Tyr or Phe, R³ isoptionally substituted Gly or an optionally substituted α-D-amino acidresidue and R⁹ is a protecting group, or its salt,

(10) A peptide according to the item (9), wherein R¹ is His, R² is Tyr,R³ is Gly, D—Leu, D—Trp, D—Val which may be substituted with C₁₋₄ alkyl,D—Ser, D—Ala which may be substituted with C₁₋₄ alkoxy, with naphthyl orwith 2-methylindolyl, or D—His which may be substituted with C₇₋₁₀aralkyl, R⁹ is C₁₋₆ alkyl, C₆₋₁₀ aryl or C₇₋₁₂ aralkyl, or its salt,

(11) A peptide according to the item (9), wherein R¹ is His, R² is Tyr,R³ is D—Leu, R is C₁₋₆ alkyl, or its salt,

(12) A method for producing a peptide of the formula:

5—oxo—Pro—R¹—Trp—Ser—R²—R³—OH  (II) (SEQ ID NO: 4)

wherein R¹ is His, Tyr, Trp or p—NH₂—Phe, R² is Tyr or Phe, R³ isoptionally substituted Gly or an optionally substituted α-D-amino acidresidue, or its salt, which comprises hydrolyzing a peptide of theformula:

5—oxo—Pro—R¹—Trp—Ser—R²—R³—OR⁹  (IV) (SEQ ID NO: 3)

wherein R¹, R² and R³ have the same meanings as defined above, R⁹ is aprotecting group, or its salt,

(13) A crystal of a peptide of the formula:

5—oxo—Pro—R¹—Trp—Ser—R²—R³—OH  (II) (SEQ ID NO: 4)

wherein R¹ is His, Tyr, Trp or p—NH₂—Phe, R² is Tyr or Phe, R³ isoptionally substituted Gly or an optionally substituted α-D-amino acidresidue, or its salt,

(14) A method for producing a crystal of a peptide of the formula:

5—oxo—Pro—R¹—Trp—Ser—R²—R³—OH  (II) (SEQ ID NO:4)

wherein R¹ is His, Tyr, Trp or p—NH₂—Phe, R² is Tyr or Phe, R³ isoptionally substituted Gly or an optionally substituted α-D-amino acidresidue, or its salt, which comprises subjecting a solution of thepeptide (II) or its salt to aging, and

(15) A method according to the item (14), wherein the solution of thepeptide (II) or its salt with the concentration of about 0.01 to 0.05mole/liter is subjected to aging at a temperature ranging from about 10to 70° C. for about 10 to 70 hours.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a typical flow chart showing a preferred process of thepresent invention.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The meanings of the symbols and/or abbreviations are shown below:

R¹ denotes His, Tyr, Trp or p—NH₂—Phe, and His is preferable.

R² denotes Tyr or Phe, and Tyr is preferable.

As α-D-amino acid residue in the optionally substituted α-D-amino acidresidue, mention is made of D—Leu, D—Ile, D—Nle, D—Val, D—Nva, D—Ser,D—Abu, D—Phe, D—Phg, D—Thr, D—Met, D—Ala, D—Trp or α-Aibu. As theα-D-amino acid residue, D—Leu, D—Val, D—Ser, D—Trp, D—Ala, D—Abu andα-Aibu are preferable, and D—Leu is more preferable.

As the substituent on the Gly or α-D-amino acid residue, mention is madeof (1) mono-C₁₋₄ alkyl, e.g. methyl, ethyl, n-propyl, isopropyl,n-butyl, t-butyl, (2) di C₁₋₄ alkyl, e.g. dimethyl, diethyl, (3) triC₁₋₄ alkyl, e.g. trimethyl, triethyl, (4) C₁₋₄ alkoxy, e.g. methoxy,ethoxy, n-propoxy, isopropoxy, n-butoxy, t-butoxy, (5) C₆₋₁₀ aryl, e.g.phenyl, naphthyl, (6) C₇₋₁₀ aralkyl, e.g. benzyl, phenethyl, (7)indolyl, (8) methylindolyl and (9) benzylimidazolyl. Among others,methyl, dimethyl, trimethyl, t-butyl, t-butoxy, 2-naphtyl, indol-3-yl,2-methylindolyl and benzylimidazol-2-yl. Especially, trimethyl, t-butyl,t-butoxy, 2-naphthyl, indol-3-yl, 2-methylindolyl andbenzylimidazol-2-yl are more preferable.

As preferable example of R³, mention is made of Gly, D—Leu, D—Trp, D—Valwhich may be substituted with C₁₋₄ alkyl, D—Ser, D—Ala which may besubstituted with C₁₋₄ alkoxy, with naphthyl or with 2-methylindolyl, orD—His which may be substituted with C₇₋₁₀ aralkyl.

As more preferable examples of R³, mention is made of Gly, D—Leu, D—Trp,3-methyl-D—Val, D—Ser, t-butoxy-D—Ala, 2-naphthyl-D—Ala,2-methylinolyl-D—Ala, benzylimidazol-2-yl—D—Ala (=N^(im)-benzyl-D—His).

R⁴ denotes Leu, Ile or Nle. As R⁴, Leu is preferable.

As the protecting group in the protected Arg of R⁵, mention is made ofan alkoxybenzenesulfonyl group, a trialkylbenzenesulfonyl group.

The alkoxybenzenesulfonyl is preferably a C₁₋₆ alkoxy-substitutedbenzenesulfonyl group such as p-methoxybenzenesulfonyl,p-ethoxybenzenesulfonyl, p-isopropoxybenzenesulfonyl, etc. and morepreferably is p-methoxybenzenesulfonyl.

The alkyl moieties in the trialkylbenzenesulfonyl group are preferablyC₁₋₆ alkyl groups, same or different, such as methyl, ethyl, propyl,n-butyl, t-butyl, n-pentyl, t-pentyl, etc. The three alkyl groups may bepresent in optional substitutable positions on the benzene ring ofbenzenesulfonyl but are preferably situated in the 2-, 4-, and6-positions with respect to the sulfonyl group. Specifically,2,4,6-trimethylbenzenesulfonyl, 2,4,6-triethylbenzenesulfonyl,2,4,6-tripropylbenzenesulfonyl, 2,4,6-triisopropylbenzenesulfonyl,2,4,6-tri—t-butylbenzenesulfonyl, etc. can be typically mentioned.

As more preferable examples of the protecting group in the protected Argof R⁵, mention is made of C₁₋₆ alkoxybenzenesulfonyl group, morepreferably p-methoxybenzenesulfonyl, p-ethoxybenzenesulfonyl,p-propoxybenzenesulfonyl, p-isopropoxybenzenesulfonyl, still morepreferably p-methoxybenenesulfonyl.

The alkyl group of the “alkyl which may optionally have hydroxyl”mentioned for the group R⁷ and R⁸ is a C₁₋₄ alkyl group such as methyl,ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl or tert-butyl,and is preferably ethyl, which may have hydroxyl in any substitutableposition.

As preferable example of the alkyl which may optionally have hydroxyl,mention is made of hydroxymethyl, 2-hydroxyethyl, 3-hydroxy-n-propyl,4-hydroxy-n-butyl, etc, and 2-hydroxyethyl is still more preferable.

As the protecting group of R⁹, mention is made of C₁₋₆ alkyl, e.g.methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl,n-pentyl, n-hexyl, etc, C₇₋₁₀ aralkyl, e.g. benzyl, phenethyl, etc. Thegroup R⁹ is preferably C₁₋₃ alkyl, more preferably ethyl.

Throughout this specification (and appended claims), amino acids andpeptides are referred to by the abbreviations according to IUPAC-IUBCommission on Biological Nomenclature or by the trivial names which arein routine use in the art. Where any amino acid may exist as opticalisomers, the L-compound is meant unless otherwise indicated.

It should also be understood that the following abbreviations, amongothers, are sometimes used in this specification.

Gly : glycine

Ala alanine

Val : valine

Leu leucine

Ile isoleucine

Ser serine

Thr : threonine

Arg : arginine

Phe : phenylalanine

Tyr : tyrosine

His : histidine

Trp : tryptophan

Pro : proline

NLe : norleucine

NVa : norvaline

Abu : 2-aminobutyric acid

Phg : phenylglycine

α-Aibu: α-aminoisobutyric acid

P—NH₂—Phe: p-aminophenylalanine

Z : benzyloxycarbonyl

Pd : palladium

Pd—C : palladium-carbon

Et : ethyl

AcOH : acetic acid

HF : hydrogen fluoride

HBr : hydrogen bromide

DCHA : dicyclohexylamine

DMF : N,N-dimethylformamide

DMA : N,N-dimethylacetamide

THF : tetrahydrofuran

MSA : methanesulfonic acid

MBS : p-methoxybenzenesulfonyl

DCC : N,N′-dicyclohexylcarbodiimide

HONB : N-hydroxy-5-norbornene-2,3-dicarboximide

HOSu : N-hydroxysuccinimide

HOBt : 1-hydroxybenzotriazole

EDA : ethyl-3-(3-dimethylamino)propylcarbodiimide hydrochloride

NP : p-nitrophenyl

HPLC : High performance liquid chromatography

Methods for the production of the peptide (I) are described below:

A variety of synthetic routes can be contemplated for the production ofthe peptide of the formula (I) or its salt and a diversity ofcombinations are available in regard to amino- and carboxy-protectinggroups, methods for amide bond formation, methods for deprotection, andmethods for purification of peptides obtained in the respective routesof synthesis.

Among such alternatives the synthetic route of FIG. 1 is one ofpreferred examples. Thus, the production technology of the presentinvention comprises (1) protecting the guanidino group of Arg⁸ with aprotecting group, (2) synthesizing two key intermediate fragments, e.g.Peptide (II) and Peptide (III), and (3) de-protecting the precursorpeptide (I′), which is available upon condensation of said peptides fromthe guanidino group of Arg⁸ to provide the objective peptide (I) or saltthereof.

As the salt of the present peptide, mention is made of salts with anorganic acid, e.g. formic acid, acetic acid, propionic acid, lacticacid, glucollic acid, pyroracemic acid, oxalic acid, malonic acid,succinic acid, maleic acid, fumaric acid, p-toluenesulfonic acid,trifluoroacetic acid, methanesulfonic acid, phosphoric acid, etc, aninorganic acid, e.g. hydrochloric acid, hydrobromic acid, sulfuric acid,etc.

1. Method for producing Peptide (a)

The peptide of the formula (a), whose structure being shown asZ—R²—R³—OR⁹, can be produced by a manner described in “PeptideGousei-no-Kiso-to-Jikken” (Basis and Experiment of Peptide Synthesis),edited by Nobuo Izumiya et al., Maruzen Publishers, Japan; or “ThePeptide”, vol. 1, pages 76-136, Ebehard Schroder and Klaus Lubke, or ina similar manner thereto.

2. Method for producing Peptide (b) from Peptide (a)

In the production process of the present invention, peptide (b),Z—Ser—R²—R³—OR⁹ which is shown in FIG. 1, is synthesized by condensingthe dipeptide (a) with Z-Ser.

At first, the peptide (a) is subjected to a reaction of elimination ofthe group Z to produce de—Z peptide (a) (peptide (a) minutes “Z” group).As the elimination reaction, catalytic reduction using a catalyst suchas Pd, Pd—C, and HBr/AcOH treatment are preferable.

As the solvent in the catalytic reduction, alcohols, e.g. methanol,ethanol, isopropanol, n-propanol, n-butanol, t-butanol, etc, ethers,such as THF, dioxane, etc amides, such as DMF, DMA, etc, are mentioned.Among others, DMF, DMA or THF is used advantageously, becauseconcentration is unnecessary in pre-stage of the next reaction step. Thereaction temperature is about 0 to 50° C., preferably about 20 to 40° C.The reaction time is about 3 to 15 hours, preferably about 5 to 10hours. This reaction is preferably conducted under a normal pressure.

Alternatively, in order to decrease the occurrence of di-ketopiperazineat the reduction reaction, it is preferable to subject de—Z peptide (a)to a protonation reaction before the catalytic reduction. At theprotonation reaction, an acid is added to the reaction system. As theacid, mention is made of inorganic acid or organic acid, preferablyhydrochloric acid, sulfuric acid, p-toluenesulfonic acid. Among them,p-toluenesulfonic acid is more preferable. The amount of the acid ispreferably about 0.8 to 1.5 times (mole/mole), more preferably 1 to 1.1times (mole/mole), that of de—Z peptide (a).

The HBr/AcOH treatment is carried out by reacting Peptide (a) with HBrtogether with a saturated acetic acid without any solvent. The reactiontemperature is about −10 to 30° C., preferably about 10 to 20° C. Thereaction time is about 10 minutes to 2 hours, preferably about 30minutes to one hour. The recovery of the objective peptide from thereaction mixture is carried out by recovering the emerged precipitate byadding ether, ethyl acetate and so on, and then by drying.

In the reaction of the introduction of Z—Ser group, the active estermethod is used, i.e. converting the peptide to an active ester, and, theintramolecular dehydration reaction is successfully inhibited byoptimization of reaction temperature. The active ester that can be usedfor the introduction of Z—Ser in the process of the invention includesvarious active esters that can be used in peptide synthesis, such as theesters with an active esterification agent, e.g.N-hydroxy-5-norbornene-2,3-dicarboximide (HONB), N-hydroxysuccinimide(HOSu), N-hydroxybenzotriazole (HOBt), etc. Particularly preferred isthe HONB ester.

For preparing an active ester of Z—Ser—OH, Z—Ser—OH is reacted with anactive esterification agent in a solvent such as ethers, e.g.diethylether, isopropylether, THF, dioxane, etc, amides, e.g. DMF, DMA,etc, acetonitrile, acid esters, e.g. methyl formate, methyl acetate,ethyl acetate, etc, at a temperature ranging from about −5 to 20° C.,preferably about 0 to 5° C., for about 5 to 15 hours, preferably about 7to 10 hours;

The reaction of de—Z peptide (a) an active ester of and Z—Ser—OH iscarried out in an inert solvent. The solvent used in the reductionreaction in the former step may be used. Among others, amides, e.g. DMF,DMA, are preferable. The reaction is carried out at a lower temperaturesuch as a temperature ranging from −10 to 10° C., preferably about −5 to5° C., for about 5 to 20 hours, preferably about 7 to 12 hours.

3. Method for producing Peptide (c) from Peptide (b)

Peptide (c), Z—Trp—Ser—R²—R³—OR⁹ (SEQ ID NO: 1) which is shown in FIG.1, is produced by subjecting a peptide obtained by de—Z reaction ofPeptide (b) (de—Z peptide (b)) with a peptide, Z—Trp—OH to acondensation reaction.

The de—Z reaction is carried out by a catalytic reduction by employing acatalyst such as Pd, Pd—C, or by a treatment using HBr/AcOH treatment.The catalytic reduction and HBr/AcOH treatment is carried out by amanner similar to those mentioned in the method for producing Peptide(b) from Peptide (a).

Introduction of Z—Trp into the peptide obtained by de—Z reaction ofPeptide (b) can be carried out by reacting Peptide (b) with an activeester of Z—Trp—OH, which is previously produced by reacting Z—Trp—OHwith an active esterification agent, e.g. HONB, HOBt, HOSu, in thepresence of a condensation agent, e.g. DCC, EDA, etc. Also, Peptide (b)and Z—Trp—OH can be condensed by reacting both using a condensationagent in the presence of an active ester.

As the active ester, HONB is preferable, and as the condensation agent,DCC is preferable. The method for producing active ester of Z—Trp—OH canbe a similar manner as above.

The reaction of Peptide (b) with the active ester of Z—Trp—OH in thepresence of a condensation agent is carried out at a temperature rangingfrom about 0 to 20° C., preferably about 5 to 10° C. for about 5 to 20hours, preferable about 7 to 10 hours.

4. Method for producing Peptide (e) from Peptide (c) and Peptide (d)

Peptide (d), Z-5-oxo—Pro—R¹—OH, can be produced by the method ofHatanaka et al. [Takeda Research Report, 35, 16 (1976); Biochemical andBiophysical Research Communications, 60, 1345 (1974)], or a similarmethod as above.

Peptide (e), Z-5-oxo—Pro—R¹—Trp—Ser—R²—R³—OR⁹ (SEQ ID NO:2), is producedby a condensation reaction of Peptide (c) with Peptide (d). In thecondensation, attention must be paid to the isomerization of the Hisresidue but any of the known anti-isomerizing procedures available forthe fragment condensation of peptides can be utilized. A typicalprocedure for suppressing this isomerization consists in the use of ananti-isomerizing agent in addition to a condensing agent. The condensingagent may for example be DCC or EDA and the anti-isomerizing agent mayfor example be HONB, HOSu, HOBt, (HOSu), or the like. These two kinds ofagents can be used in any desired combination. Typical combinations areDCC—HONB, DCC—HOSu, DCC—HOBt, and EDA—HOSu, among others. Particularlypreferred is DCC—HONB. While the reaction temperature is a major factorin the control of isomerization, the preferred reaction temperature isabout 0 to 20° C., preferably about 5. to 15° C. for 30 minutes to 100hours, preferably about 50 to 80 hours. In the reaction, as a solvent,use is made of amides, e.g. DMF, DMA, etc, ethers, e.g.2-methylpyrrolidone, THF, dioxane, etc.

5. Method for producing Peptide (IV) from Peptide (e)

Elimination of Z from Peptide (e), Z-5-oxo—Pro—R¹—Trp—Ser—R²—R³—OR⁹ (SEQID NO: 2), gives Peptide (IV). The de—Z reaction is carried out withmethods such as catalytic reduction with a catalyst, e.g. Pd, Pd—C, orthe like, or HBr/AcOH treatment.

In the catalytic reduction, as a solvent, use is made of amides, e.g.DMF, DMA, etc, ethers, e.g. 2-methylpyrrolidone, THF, dioxane, etc, ort-butanol, at a temperature ranging from about 0 to 50° C., preferablyabout 25 to 40° C., for about 1 to 10 hours, preferably about 3 to 6hours.

The HBr/AcOH treatment is carried by a method similar to those mentionedin the reaction from Peptide (a) to Peptide (b).

6. Method for producing Peptide (II) from Peptide (IV)

Peptide (II) can be produced by hydrolyzing Peptide (IV), and ifnecessary, neutralizing the reaction mixture.

This hydrolysis reaction is preferably carried out in a mixture of waterand an alcohol at a low temperature. The alcohol that can be usedincludes methanol, ethanol, propanol, n-butanol, etc. and the proportionof the alcohol in the water-alcohol mixture is about 1 to 30% (v/v),preferably about 1 to 10% (v/v). The larger the proportion of thealcohol, the lower is the velocity of hydrolysis reaction and, at thesame time, the rates of isomerization of His² and Ser⁴ are significantlyincreased (when R¹ is His). These isomers cannot be efficiently removedeven by the column chromatographic system used in purification, thusdetracting from the quality of the final product, Peptide (I).

The reaction solvent may contain the solvent carried over from theupstream stage, viz. DMF, THF, or the like, but its proportion shouldnot be large enough to retard the reaction in any appreciable measure,i.e. preferably about 1 to 20% (v/v), preferably about 5 to 10% (v/v).

The hydrolysis is carried out in the presence of alkali. As the alkali,mention is made of sodium hydroxide, potassium hydroxide, calciumhydroxide, barium hydroxide. The reaction speed of hydrolysis andisomerization of His at 2-position and Ser at 4-position depend upon theamount of alkali and its concentration in the solution. The amount ofthe alkali is preferably about 2.5 to 2 times mole/mole per Peptide(IV), preferably about 3 to 4 times mole/mole. In particular, theconcentration of alkali in the reaction system at the initial time ofthe reaction is preferably about 0.05 to 0.3 mole/liter, more preferablyabout 0.1 to 0.2 mole/liter. The reaction temperature should bemaintained at about −10 to 10° C., preferably about −5 to 5° C., up tillcompletion of the after-treatment, namely neutralization. In order tokeep the reaction temperature constant, the reaction system ispreferably agitated. This reaction generally goes to completion in 1.5to 3 hours. In order that the objectionable isomerization may beminimized, the neutralization procedure is preferably carried outimmediately following the reaction. This neutralization is carried outat a temperature ranging from −5 to 5° C., preferably −2 to 2° C. usingan acid (e.g. hydrochloric acid, sulfuric acid, etc.).

The peptide (II) can be crystallized by the following method:

Thus, the gels formed in the neutralization of the reaction mixturefollowing hydrolysis of (IV) are dissolved by heating and then allowedto cool gradually to let Peptide (II) crystallize out. The dissolutionby heating is carried out at a temperature ranging from about 60 to 80°C., preferably about 65 to 75° C. under stirring. This crystallizationcan be faciliated by adding seed crystals in the cooling phase. As tothe timing of addition, seed crystals can be added at any time duringthe phase where the added seed crystals will not dissolve in thesolution and the hot solution will not give rise to gels again.Preferred is the phase where the temperature of the solution is about 35to 45° C. The gradual cooling is carried out for about 1 to 3 hours, ata temperature ranging from 15 to 30° C.

In order to complete the crystallization, it is preferable to subjectthe product to aging. The time for aging for obtaining crystals ofPeptide (II), which have excellent properties with a high yield, isabout 30 to 150 hours, preferably about 60 to 100 hours. The temperatureof aging is preferably about 10 to 35° C., more preferably about 10 to30° C., still more preferably about 15 to 25° C.

As the concentration of Peptide (II) in the solution at the initial timeof aging, about 0.01 to 0.05 mole/liter is provided, preferably about0.02 to 0.04 mole/liter.

In the course of aging, it is preferable to stir the Peptide(II)-containing solution intermittently, for shortening the aging timeand/or obtaining a high yield of the crystals. Thus it is preferable tostir the solution for about one minute at about 30 to 35 rpm commencingfive hours into the reaction. Thereafter, the solution is stirred forabout one minute at about 30 to 35 rpm about every five hours until theaging process is ⅔ completed. Thereafter, the solution is stirred forabout one minute at about 30 to 35 rpm about every 0.5 hours until thefinal step of aging.

More concretely, e.g., the aging time is 60 hours, the solution isstirred at 33 rpm for 1 minute every 5 hours beginning 5 hours into thereaction, until 40 hours. Thereafter, the solution is stirred at 33 rpmfor 1 minute every 0.5 hour from 40 hours after the starting of theaging to the last step.

By the above crystallization procedure for Peptide (II), thetime-consuming operations for removal of contaminants that would affectthe final product quality, such as the His isomer by-produced in thefragment condensation reaction of Peptide (c) to Peptide (e) andhydrolysis reaction of Peptide (IV) to Peptide (II) and the Ser isomerby-produced in the hydrolysis reaction of Peptide (IV) to Peptide (II),as well as the procedure for isolation of Peptide (IV), can all beomitted.

The Peptide (II) can be used for the next reaction in the form of a saltwith an alkali metal, e.g. Li, Na, K, Ca, Ba, etc., or an organic base,e.g. triethylamine, cyclohexylamine, dicyclohexylamine, either asisolated by a known method such as crystallization or in the solutionform as such.

7. Production of Peptide (III)

Peptide (III) or a salt thereof can be produced by the method of Fujinoet al. Archives of Biochemistry and Biophysics, 154, 488 (1973);Chemical and Pharmaceutical Bulletin, 23, 229 (1975)] or any other knownmethod for peptide synthesis.

Peptide (III) can be used for the next reaction in the form of a saltwith, for example, hydrochloric acid, hydrobromic acid, sulfuric acid,p-toluenesulfonic acid, trifluoroacetic acid, or methanesulfonic acid,either as isolated by a known separation procedure such ascrystallization or in the solution form as such. In such cases, however,the amide bond-forming reaction has to be preceded by elimination of thebase (acid) by neutralization with a base or treatment with an ionexchange resin.

8. Production of Peptide (I′) from Peptide (II) and Peptide (III)

The condensation reaction of Peptide (II) with Peptide (III) to givePeptide (I′) is carried out in a solvent which does not interfere withthe reaction. The solvent that can be used includesN,N-dimethylformamide (DMF), N,N-dimethylacetamide (DMA),N-methylpyrrolidone, dichloromethane, dichloroethane, tetrahydofuran,dioxane, etc. and these solvents can be used as a suitable mixture.Preferred are N,N-dimethylformamide and N,N-dimethylacetamide.

The proportion of Peptide (III) relative to Peptide (II) is about 0.5 to2 molar equivalents, preferably about 1 to 1.5 molar equivalents.

The reaction temperature is generally about 0 to 40° C. and preferablyabout 5 to 25° C. The range of the temperature is important forpreventing isomerization of R³. The reaction time is generally about 30to 60 hours and preferably about 40 to 50 hors.

In the condensation reaction of Peptide (c) with Peptide (d), attentionmust be paid to the isomerization of the group R³ but any of the knownanti-isomerizing procedures available for the fragment condensation ofpeptides can be utilized. A typical procedure for suppressing thisisomerization (racemization) consists of the use of an anti-isomerizingagent in addition to a condensing agent. The condensing agent may forexample be DCC or EDA and the anti-isomerizing agent may for example beHONB, HOSu, HOBt, or the like. These two kinds of agents can be used inany desired combination. Typical combinations are DCC—HONB, DCC—HOSu,DCC—HOBt, and EDA—HOSu, among others. Particularly preferred isDCC—HONB.

The condensing agent is used at a ratio of one to 3 times (mole/mole) ofPeptide (II), preferably one to two times (mole/mole). The amount ofanti-isomerizing agent is one to 4 times (mole/mole) of Peptide (II),preferably 1.5 to 2.5 times (mole/mole). The initial concentration ofPeptide (II) is about 0.05 to 0.2 mole/liter, preferably 0.08 to 1.5mole/liter. Peptide (III) is used in an amount of 0.8 to 2 times(mole/mole), preferably 1 to 1.3 times (mole/mole), relative to Peptide(II).

9. Production of peptide (I) from Peptide (I′)

The deprotection reaction for removing the protective group from the Argresidue of Peptide (I′) to give Peptide (I) can be carried out with anacid in a solvent that does not interfere with the reaction or in theabsence of a solvent. The solvent that does not interfere with thisdeprotection reaction includes but is not limited to dichloromethane,dichloroethane, dioxane, and trifluoroacetic acid, and these solventscan be used as a suitable mixture.

The acid that can be used for this deprotection reaction includes but isnot limited to C₁₋₆ alkanesulfonic acids, e.g. methanesulfonic acid,ethanesulfonic acid, etc., halogenosulfonic acids, e.g. chlorosulfonicacid, fluorosulfonic acid, bromosulfonic acid, etc, and Lewis acids,e.g. boron tris(trifluoroacetate), etc. Among these, C₁₋₃ alkanesulfonicacids are preferred and methanesulfonic acid is particularly preferred.

The proportion of the acid for use in this deprotection procedure isabout 5 to 25 (w/w) times, preferably about 10 to 20 (w/w) times, basedon the weight of Peptide (I′).

The deprotection reaction temperature is generally about 0 to 20° C. andpreferably about 5 to 15° C.

The deprotection reaction time is generally about 2 to 8 hours andpreferably about 4 to 6 hours.

This deprotection reaction is preferably carried out usingmethanesulfonic acid in the absence of a solvent.

In conducting this deprotection reaction, a radical scavenger (e.g.phenol, anisole, etc.) and/or an antioxidant (e.g. thioglycolic acid)may be added each in a suitable amount. Their amount may each be about0.8 to 2 (w/w) times, preferably about 0.05 to 2 (w/w) times, based onthe above-mentioned reaction product.

When the deprotection reaction is conducted using a C₁₋₆ alkanesulfonicacid, the alkanesulfonic acid can be eliminated from the reactionmixture by, for example, the method comprising washing the reactionmixture with ether, dissolving water in the washes, and running theresulting solution on an anion exchange resin column. However, when thismethod is applied to commercial-scale production, the risk of using alarge quantity of ether because the solubility of C₁₋₆ alkylsulfonicacid in ether is very low, the risk of abrupt evolution of heat at themixing of a large quantity of the alkanesulfonic acid with water andupon running the solution on the ion exchange resin column, and thedecomposition of the objective peptide due to the evolved heat make themethod unsuitable from the standpoints of hardware required,workability, and safety.

As a commercially advantageous method free of the above problems, thereis a technology for eliminating the alkanesulfonic acid from thereaction mixture by direct neutralization with a basic aqueous medium.However, in this neutralization process involving neutralization of alarge quantity of the strong acid, evolution of an intense heat ofneutralization is naturally expected despite the advantage offeasibility of large-scale treatment. In addition to the problem that alarge quantity of the neutralized solution must be handled, the adverseinfluences on the quality of the end product compound due to cleavage ofpeptide bonds and isomerization of amino acid residues of the objectivepeptide in the basic aqueous medium are inevitable so that the methodwas considered to be lacking in common sense as far as the production ofpeptides is concerned.

However, the inventors of the present invention explored into themethodology for absorbing the heat of neutralization evolved in thisneutralization reaction as well as the procedural aspect of themethodology and made efforts to optimize the reaction conditions. As aresult, they discovered surprisingly that the objective compound ofacceptable quality could be obtained in good yield and have establisheda production-scale method for said neutralization. Thus, in accordancewith the present invention, the reaction mixture is added dropwise to acooled aqueous solution of an inorganic or organic base to neutralizethe alkanesulfonic acid at a sustained low temperature. In this mode ofoperation, isomerization of constituent amino acid residues of theobjective peptide can be successfully inhibited and the objectivecompound be obtained either as solid or as oil.

The inorganic base that can be used for this purpose includes sodiumhydroxide, potassium hydroxide, calcium hydroxide, sodium carbonate,potassium carbonate, calcium carbonate, etc., and the organic base thatcan be used includes pyridine, triethylamine, etc., although potassiumcarbonate is particularly advantageous. When a carbonate is used as saidbase, copious foaming due to evolution of carbon dioxide gas isinevitable but this foaming can be controlled by adding an organicsolvent such as ethyl acetate or benzene in a suitable proportionbeforehand. The amount of said inorganic or organic base need only besufficient to neutralize the alkanesulfonic acid but is preferably about1 to 1.3 equivalents relative to the alkanesulfonic acid.

In order that the isomerization of amino acid residues and hydrolyticcleavage of the peptide chain may be inhibited and freezing of saidaqueous solution of inorganic or organic base may be prevented, theneutralization system temperature is preferably controlled at about −15to 15° C., preferably about −5 to 5° C.

Upon neutralization, the objective compound usually separates out assolid or oil from the neutralization system and, therefore, can berecovered by a suitable known procedure such as filtration ordecantation.

The objective compound separated from the neutralization system can bepurified by per se known procedures. In the purification of a relativelysmall amount of the objective compound, liquid chromatographyconstitutes a method of choice. For the purification of a large amountof the objective compound, there can be employed a combination ofseveral known column chromatographic systems according to the ionicnature, polarity and other solution properties, aromaticity, andmolecular weight of the particular compound.

The fractional purification technology preferred from industrial pointsof view includes a judicious combination of column chromatographicsystems using a hyperporous resin such as Amberlite XAD-2 (Rohm & HaasCo., USA) or Diaion HP-20 (Mitsubishi Chemical, Japan),carboxymethylcellulose (CMC, CM-23 (Whatman, USA)), an ion exchangeresin such as Amberlite CG-50 (Rohm & Haas Co., USA), and a molecularsieve resin such as Sephadex LH-20 (Pharmacia Fine Chemicals, Sweden).This procedure is procedurally simple and safe and provides for goodreproducibility of both yield and quality, besides being economical.

An exemplary combination of column chromatographic systems may be aserial combination of Diaion HP-20 (mentioned above) (lst run)—CM-23(mentioned above)—Diaion HP-20 (2nd run) - Sephadex LH-20 (mentionedabove). When a solution containing a peptide is subjected toconcentration, it emerges vigorous foaming, and it is necessary to use alyophilizing machine, or to add an anti-foaming agent. However, thecombination of said column chromatography brings an advantageous methodthat the concentration procedure is omitted, the concentration beingthat on the effective fraction obtained in each column chromatography,especially the concentration of a large amount of the aqueous solutioncontaining the effective fraction obtained by column chromatography ofCM-23. Therefore, the combination of the above chromatography providesan industrially advantageous method in terms of operability andeconomically, as well as high quality which depend upon restrainingdecomposition of peptides due to concentration.

Diaion HP-20 (1st run) column chromatography has for its primary objectto eliminate inorganic contaminants, as well as the radical scavengeradded in the deprotection step and its reaction product, the antioxidantoptionally added, and isomers of the objective compound as produced inminor amounts, from the solid or oil containing the crude objectivecompound as separated from the reaction mixture after the deprotectionreaction. Referring, further, to this Diaion HP-20 (lst run) columnchromatography, Diaion HP-20 is used in a proportion of about 20 to 40(v/w) times, preferably about 25 to 35 times, relative to the objectivecompound. Elution of the objective compound and contaminants isgenerally carried out with an aqueous solution of acetone, methanol, orethanol. In the following exemplary description, ethanol is used. First,the aqueous solution containing the objective compound is run on aDiaion HP-20 column and the column is irrigated serially with programmedamounts of sodium acetate/water preadjusted to pH 5 to 7 with aceticacid, ammonium acetate/water, and 10% (v/v) ethanol to elute relatedcompounds. Then, the objective compound is eluted with 15% (v/v) ethanoland 35% (v/v) ethanol and the programmed fractions are pooled.

CM-23 column chromatography has for its primary object to eliminatebyproducts tonically non-equivalent to the objective compound, and CM-23is used in a proportion of generally 35 to 60 times (vlw), preferably40-55 times (v/w), relative to the objective compound. The solutionavailable after distillation of ethanol from the programmed eluate fromthe Diaion HP-20 (1st run) column is run on a CM-23 column and thecolumn is first rinsed with water. The objective compound is then elutedby serial elution with 0.015 M ammonium acetate/water and 0.03 Mammonium acetate/water and the programmed fractions are pooled.

Diaion HP-20 (2nd run) column chromatography has for its primary objectto remove the ammonium acetate used in elution from the CM-23 column andconcentrate the large amount of eluate. The programmed eluate availableafter CM-23 column chromatography (a large quantity of aqueous solutionwhich would give copious foaming on concentration) can be applieddirectly, i.e. without prior concentration, to the Diaion HP-20 (2ndrun) column. This Diaion HP-20 (2nd run) column is irrigated seriallywith sodium acetate/water preadjusted to pH about 5 to 7 with aceticacid, ammonium acetate/water, and water in the order mentioned. Theobjective compound is then eluted with 15% (v/v) ethanol and 35% (v/v)ethanol and the programmed fractions are pooled. The programmed eluatethus obtained from the Diaion HP-20 (2nd run) column (which has beenconcentrated to about ⅓ by volume of the programmed eluate from theCM-23 column) is concentrated under reduced pressure and the residue isrun on a Sephadex LH-20 column.

Sephadex LH-20 column chromatography, which is the final stage of thecascade, has for its object to eliminate pyrogenic substances, inorganicmatter, and other trace contaminants. Sephadex LH-20 is used in aproportion of about 20 to 60 times (v/w), preferably about 30 to 50times (vi/w), relative to the objective compound. The LH-20 column isdeveloped with 0.005N acetic acid/H₂O and the programmed fractions arepooled. Where necessary, this programmed eluate is concentrated, treatedwith active charcoal, membrane-filtered, and lyophilized to give theobjective compound (acetate) as a final product.

Thus obtained Peptide (I) has a LHRH agonist activity, and can be used,for example, a similar manner as described in U.S. Pat. No. 4,008,209.

EXAMPLES

The following examples are intended to describe the present inventionmerely in further detail and should by no means be construed as definingthe scope of the invention.

Reference Example 1

Production of Z—Tyr—D—Leu—OEt

Z—TyrOH•DCHA (58.8 g) was desalted with 1N-sulfuric acid in about 300 mlof ethyl acetate at 0 to 10° C. The organic layer was separated anddehydrated over anhydrous sodium sulfate (Na₂SO₄). After the Na₂SO₄ wasfiltered off, 24.3 g of D—Leu—OEt, 12.6 g of triethylamine, and 26.8 gof DCC were added to the filtrate and the mixture was stirred at about5° C. for about 2 hours and further at about 10° C. for 5 hours. To thisreaction mixture was added 60 ml of 1 N-hydrochloric acid and themixture was filtered. The filtrate was allowed to stand and the organiclayer was washed with aqueous solution of NaCl and aqueous solution ofNaHCO₃ and the ethyl acetate was distilled off. To the residue was addedisopropyl ether and the resulting crystal crop was harvested byfiltration, recrystallized from ethyl acetate-isopropyl ether, anddried.

Yield 41.6 g (77%)

m.p. 116-118° C.

Optical rotation [α]_(D) ²⁵=−3.4° (c=1, DMF)

Reference Example 2

Production of Z—Ser—Tyr—D—Leu—OEt

In about 300 ml of DMF were dissolved 40.5 g of Z—Tyr—D—Leu—OEt and 16.9g of p-toluenesulfonic acid monohydrate, and hydrogenation was carriedout in the presence of about 5 g of 5% Pd—C at 20 to 35° C. Aftercompletion of the reaction, the catalyst was filtered off.

Separately, 23.3 g of Z—Ser—OH and 19.2 g of HONB were dissolved in 300ml of DMF followed by addition of 22.1 g of DCC and the mixture wasstirred at 0 to 5° C. for about 7 hours. To this mixture was added theabove reduction reaction mixture. The mixture was cooled to about 0° C.and 9 g of triethylamine was added dropwise. The mixture was thenstirred at −5 to 5° C. for about 10 hours and left standing at roomtemperature overnight.

The crystals which formed were filtered off and the filtrate wasconcentrated under reduced pressure. The residue was dissolved in ethylacetate. This ethyl acetate solution was washed with 1 N—HCl, aqueoussolution of NaCl, and aqueous solution of NaHCO₃ in that order, anddehydrated over Na₂SO₄. After the Na₂SO₄ was filtered off, the filtratewas concentrated under reduced pressure and diluted with isopropylether. The resulting crude crystal crop was harvested by filtration,recrystallized from ethyl acetate, and dried.

Yield 37.6 g (77%)

m.p. 134-136° C.

Optical rotation [α]_(D) ²⁵=−4.8° (c=1, DMF)

Reference Example 3

Procution of Z—Trp—Ser—Tyr—D—Leu—OEt (SEQ ID NO: 7) (ETSTLE)

In 300 ml of DMF was dissolved 36.2 g of Z—Ser—Tyr—D—Leu—OEt, andhydrogenation was carried out in the presence of about 5 g of 5% Pd—C at25 to 35° C. After completion of the reaction, the catalyst was filteredoff.

Separately, 21.4 g of Z—Trp—OH and 11.9 g of HONB were dissolved inabout 300 ml of DMF followed by addition of 13.7 g of DCC and themixture was stirred at 5 to 10° C. for about 7 hours. To this mixturewas added the above reduction reaction mixture. The mixture was thenstirred at 10 to 15° C. for about 7 hours and left standing at roomtemperature overnight.

The crystals which formed were filtered off and the filtrate wasconcentrated under reduced pressure. The residue was dissolved in ethylacetate and this solution was washed with 1 N—HCl, aqueous solution ofNaCl, and aqueous solution of NaHCO₃ in that order, and dehydrated overNa₂SO₄. After the Na₂SO₄ was filtered off, the filtrate was concentratedunder reduced pressure and isopropyl ether was added to the residue. Theresulting crude crystal crop was harvested by filtration, recrystallizedfrom ethyl acetate-isopropyl ether, and dried.

Yield 41.3 g (85%)

Optical rotation [α]_(D) ²⁵=−10.0° (c=1 , EtOH)

Reference Example 4

Production of Z-5—oxo—Pro—His—Trp—Ser—Tyr—D—Leu—OEt (SEQ ID NO: 8)

In about 350 ml of DMF was dissolved 55.7 g of Z—Trp—Ser—Tyr—D—Leu—OEt(ZTSTLE), and hydrogenation was carried out in the presence of about 12g of 5% Pd—C (wet) at about 30° C. After completion of the reaction, thecatalyst was filtered off. To the filtrate thus obtained were added 32.6g of Z-5-oxo—Pro—His—OH•1.5 H₇O and 27.4 g of HONB, and after cooling to2 to 8° C., 20.5 g of DCC was added. The mixture was stirred at about 8°C. for about 60 hours.

This reaction mixture was further stirred at about 40° C. for about 2hours and then filtered and the filtrate was concentrated under reducedpressure. To the residue was added about 1 L of ethyl acetate at about60° C. and the mixture was stirred at 20 to 25° C. The resulting crudecrystals were collected by filtration, suspended in DMF (ca 230ml)-ethyl acetate (ca 530 ml), stirred, and recovered by filtration. Thepurified crystals (ZPGLE) thus obtained were not dried but directlysubmitted to the next reduction reaction.

Reference Example 5

Synthesis of 5-oxo—Pro—His—Trp—Ser—Tyr—D—Leu—OEt (SEQ ID NO: 9)

The ZPGLE obtained in Reference Example 4 was dissolved in about 900 mlof DMF and hydrogenation was carried out in the presence of 15 g of 5%Pd—C (wet) at about 30° C. After completion of the reaction, thecatalyst was filtered off and the filtrate was concentrated to about 180ml and subjected to the next hydrolysis reaction.

Reference Example 6

Production of Z—Leu—ONP

In 1.2 L of ethyl acetate was dissolved 125 g of Z—Leu—OH as well as65.5 g of p-nitrophenol, followed by dropwise addition of a solution of107 g of DCC in ethyl acetate at 0 to 5° C., and the mixture was stirredat 10 to 25° C. After completion of the reaction, crystals were filteredoff and the filtrate was concentrated under reduced pressure. Theresidue was dissolved in ethanol and the crystals which formed werecollected by filtration and dried in vacuo.

Yield 142 g (78%)

m.p. 92-94° C.

Optical rotation [α]_(D) ²⁵=−42.5° (c=1, CH₃OH)

Reference Example 7

Production of Z—Arg(MBS)—Pro—NHC₂H₅

In 400 ml of ethyl acetate was suspended 34.1 g of Z—Arg(MBS)—OH—DCHA,followed by addition of 57 ml of 1 N-sulfuric acid at 0 to 10° C. Themixture was stirred and allowed to stand and the organic layer waswashed with aqueous solution of sodium sulfate and concentrated underreduced pressure. On the other hand, 15.7 g of Z—Pro—NHC₂H₅ wasdissolved in 30 ml of N,N-dimethylacetamide and hydrogenation wascarried out in the presence of 2.3 g of 5% Pd—C. After completion of thereaction, the catalyst was filtered off. In the filtrate was dissolvedthe Z—Arg(MBS)—OH-containing concentration residue as well as 9.4 g ofHONB. To this solution was added a solution of 12.8 g of DCC inN,N-dimethylacetamide dropwise and the mixture was stirred at 10 to 20°C. After completion of the reaction, the crystals which formed werefiltered off and the filtrate was concentrated under reduced pressure.The residue was dissolved in 450 ml of ethyl acetate and washed seriallywith 1 N—HCl, aqueous solution of NaCl, and aqueous solution of NaHCO₃in that order. The ethyl acetate was distilled off under reducedpressure and the residue was treated with ethyl acetate-ethanol. Theresulting crude crystalline crop was recrystallized from ethanol anddried in vacuo.

Yield 22.4 g (72%)

Optical rotation [α]_(D) ²⁵=−33.0° (c=1, CH₃OH)

Reference Example 8

Production of Z—Leu—Arg(MBS)—Pro —NHC₂H₅ (ZLAP)

In 160 ml of DMF were dissolved 20.3 g of Z—Arg(MBS)—Pro—NHC₂H₅ and 6.4g of p-toluenesulfonic acid monohydrate, and hydrogenation was carriedout in the presence of 2.3 g of 5% Pd—C. After completion of thereaction, the catalyst was filtered off and 3.4 g of triethylamine wasadded to the filtrate under ice-cooling. To this mixture was added 13.6g of Z—Leu—ONP and the mixture was stirred at 10 to 15° C. Aftercompletion of the reaction, the reaction mixture was concentrated underreduced pressure and the residue was dissolved in 120 ml of ethylacetate. This solution was washed serially with diluted hydrochloricacid, aqueous solution of NaHCO₃, and water. The ethyl acetate was thendistilled off and the residue was applied to a silica gel column. Thecolumn was developed serially with ethyl acetate-isopropyl ether (1:1)and ethyl acetate-methanol (3:2) and the objective fractions were pooledand concentrated under reduced pressure. The residue was dissolved inethyl acetate and treated with isopropyl ether and the resultingprecipitate was recovered by filtration and dried in vacuo.

Yield 22.2 g (92%)

Optical rotation [α]_(D) ²⁵=−39.0° (c=1, CH₃CH₂OH)

Example 1

Production of 5-oxo—Pro—His—Trp—Ser—Tyr—D—Leu—OH (SEQ ID NO: 10) (PGLOH)

To the concentrate obtained in Reference Example 5 was added a solutionof 8.3 g of sodium hydroxide in 1 L of water dropwise at −3 to 0° C. andthe mixture was stirred at about 0° C. for about 2 hours. After thecompletion of reaction was confirmed, the reaction mixture wasneutralized by adding 210 ml of 1 N-hydrochloric acid dropwise at about0° C. The crystals which formed were dissolved by heating and 1.5 g ofactive charcoal was added under heating. After stirring, the activecharcoal was filtered off. The filtrate was allowed to cool and stirredat 15 to 25° C. for 35 hours. The resulting crystals were collected byfiltration and dried in vacuo at about 60° C.

Yield 39.9 g (59.2%, based on ZTSTLE obtained in Reference Example 3)

Optical rotation [α]_(D) ²⁵=−21.5° (c=0.5, DMF)

(1) Incidentally, whereas the objectionable isomerization at His in2-position and Ser in 4-position after one hour of reaction using theabove solvent system were 1.3% and 4.8%, respectively, at the reactiontemperature of 20° C., the corresponding rates at the reactiontemperature of 0° C. were 0.2% and 0.44%, respectively.

(2) While the crystallization procedure according to the presentinvention effectively eliminates various structurally related compounds,examples of this elimination effect are shown below with focus on theproducts of His-isomerization and Ser-isomerization which are the mostsignificant related compounds.

TABLE 1 Reaction mixture Crystals [D-His²]compound 0.88%* 0.18%[D-Ser⁴]compound 0.44% 0.08% *Inclusive of the [D-His²] isomerby-produced in the fragment condensation step of the method for theproduction of Peptide (e) from Peptide (c).

Example 2

Production of crystals of 5-oxo—Pro—His—Trp—Ser—Tyr—D—Leu—OH (SEQ ID NO:10) (PGLOH)

To the concentrate obtained in Reference Example 5 was added a solutionof 8.3 g of sodium hydroxide in 1 L of water dropwise at −3 to 0° C. andthe mixture was stirred at about 0° C. for about 2 hours. After thecompletion of reaction was confirmed, the reaction mixture wasneutralized by adding 210 ml of 1 N-hydrochloric acid dropwise at about0° C. The crystals which formed were dissolved by heating and 1.5 g ofactive charcoal was added under heating. After stirring, the activecharcoal was filtered off. The filtrate was allowed to aging at 18 to22° C. for 80 hours. The resulting crystals were collected by filtrationand dried in vacuo at about 60° C.

Yield 42.9 g (63.6%, based on ZTSTLE obtained in Reference Example 3)

Optical rotation [α]_(D) ²⁵=−21.8° (c=0.5, DMF)

Example 3

Production of 5-oxo—Pro—His—Trp—Ser—Tyr—D—Leu—Leu—Arg(MBS)—Pro—NHC₂H₅(SEQ ID NO: 11) (briefly, MBSTAP)

In 350 ml of DMF was dissolved 35.6 g of ZLAP, and hydrogenation wascarried out in the presence of 7.3 g of 5% Pd—C. Upon completion of thereaction, the catalyst was filtered off and 36.9 g of PGLOH and 16.2 gof HONB were dissolved in the filtrate, followed by dropwise addition ofa solution of 14 g of DCC in DMF at −4 to 8° C. The mixture was stirredat about 8° C. for 15 hours and further at about 20° C. for about 30hours. After completion of the reaction, the resulting crystals werefiltered off and the filtrate was concentrated under reduced pressureand dissolved in about 200 ml of ethanol. Then, about 2.3 L of ethylacetate was added and the resulting crystalline solid was recovered byfiltration. This crude product was dissolved in about 220 ml of ethanoland about 900 ml of ethyl acetate was added. The resulting solid wasrecovered by filtration and washed with dichloromethane to provideMBSTAP as a wet product. This wet product was not dried but directlysubmitted to the next reaction for elimination of MBS. Assay by HPLCrevealed that the yield of MBSTAP was 51.1 g (82%).

Example 4

Production of5-oxo—Pro—His—Trp—Ser—Tyr—D—Leu—Leu—Arg—Pro—NHC₂H₅•CH₃COOOH (SEQ IDNO:12) (briefly, TAP-144)

(1) Elimination of MBS

In 800 g of methanesulfonic acid was dissolved 60 g of phenol, and thewet MBSTAP (equivalent to 51.1 g of MBSTAP) obtained in Example 1 wasadded to the solution under cooling. The mixture was then stirred atabout 10° C. for 5 hours.

Separately, 690 g of potassium carbonate was dissolved in 2 L of waterand mixed with about 400 ml of ethyl acetate. This mixture was cooled to−2 to 0° C. to provide an alkali solution. To this alkali solution wasadded the above de-MBS reaction mixture dropwise at about 0° C. Aftercompletion of dropwise addition, the mixture was allowed to stand and abuffer solution (pH 4) (a mixture of ca 3 L of 0.1-N sodium acetate andca. 58 ml of acetic acid) was added to the upper layer (oil-ethylacetate layer) to dissolve the oil. The mixture was then allowed tostand and the aqueous layer was washed with ethyl acetate.

The washed aqueous layer was adjusted to pH about 6 with 20% (wlw)solution of potassium carbonate and the oil that had separated out wasremoved to provide an aqueous solution of TAP-144.

(2) Purification of crude TAP-144

The above aqueous solution of TAP-144 was applied to a column (ca. 1.2L) of DIAION™ HP-20. The column was then washed serially with about 2.5L of 0.3 M sodium acetate/water (adjusted to pH 6.2 with acetic acid),about 3 L of 0.025 M ammonium acetate/water, and 4.3 L of 10% ethanol.Thereafter, 9.5 L of 15% ethanol and 9.5 L of 35% ethanol were passed inthat order and the objective fractions were pooled and concentratedunder reduced pressure to remove the ethanol (S1-TAP).

This S1-TAP was applied to a column (ca. 1.7 L) of CM™-23 and after thecolumn was washed with 2 L of water, the objective compound was elutedserially with 15 L of 0.015 M ammonium acetate/water and 15 L of 0.03 Mammonium acetate/water and the objective fractions were pooled (S2-TAP).

The S2-TAP obtained above was applied to a column of DIAION™ HP-20 (ca.0.7 L) and after the column was washed serially with about 2.1 L of 0.3M sodium acetate (adjusted to pH 6.2 with acetic acid), 3.2 L of 0.01 Mammonium acetate/water, and 0.7 L of water, elution was carried out with4.3 L of 15% ethanol and 5.4 L of 35% ethanol in that order. Theobjective fractions were pooled and concentrated under reduced pressureto about 200 ml. The residue was applied to a column of Sephadex LH-20(ca. 10 L) and elution was carried out with 0.005N-acetic acid/H₂O. Theobjective fractions were pooled and subjected to treatment with activecharcoal, ultrafiltration, concentration, and lyophilization to provide31.8 g (67.6%) of TAP-144.

Content: 99.8% (HPLC, internal standard)

Optical rotation [α]_(D) ²⁰=−39.0° (c=1, 1% acetic acid)

Absorbance: 57 (281 nm), 55 (289 nm)

The process of the present invention has many advantages: namely i) theisomerization of His² and Ser⁴ in the hydrolysis step is minimized, ii)the Peptide (II) can be obtained as quality crystals and the isomersby-produced in condensation and hydrolysis can be successfully removed,iii) the product compound available upon elimination of thealkoxybenzenesulfonyl group after treatment with an alkanesulfonic acidcan be separated on a commercial scale, iv) the final compound can bepurified on a commercial scale, v) the intermediate Peptide (b) can beobtained in good yield with the formation of byproducts beingcontrolled, vi) the drying of intermediates Peptide (e) and Peptide (I′)and the isolation of intermediate Peptide (IV) can be omitted. Thus,there is provided an industrial method for producing a peptide havingLHRH agonistic activity.

15 1 4 PRT Artificial Sequence Description of Artificial SequencePeptide 1 Xaa Ser Xaa Xaa 1 2 6 PRT Artificial Sequence Description ofArtificial Sequence Peptide 2 Xaa Xaa Trp Ser Xaa Xaa 1 5 3 6 PRTArtificial Sequence Description of Artificial Sequence Peptide 3 Xaa XaaTrp Ser Xaa Xaa 1 5 4 6 PRT Artificial Sequence Description ofArtificial Sequence Peptide 4 Xaa Xaa Trp Ser Xaa Xaa 1 5 5 9 PRTArtificial Sequence Description of Artificial Sequence Peptide 5 Xaa XaaTrp Ser Xaa Xaa Xaa Xaa Xaa 1 5 6 9 PRT Artificial Sequence Descriptionof Artificial Sequence Peptide 6 Xaa Xaa Trp Ser Xaa Xaa Xaa Arg Xaa 1 57 4 PRT Artificial Sequence Description of Artificial Sequence Peptide 7Xaa Ser Tyr Xaa 1 8 6 PRT Artificial Sequence Description of ArtificialSequence Peptide 8 Xaa His Trp Ser Tyr Xaa 1 5 9 6 PRT ArtificialSequence Description of Artificial Sequence Peptide 9 Xaa His Trp SerTyr Xaa 1 5 10 6 PRT Artificial Sequence Description of ArtificialSequence Peptide 10 Xaa His Trp Ser Tyr Xaa 1 5 11 9 PRT ArtificialSequence Description of Artificial Sequence Peptide 11 Xaa His Trp SerTyr Xaa Leu Xaa Xaa 1 5 12 9 PRT Artificial Sequence Description ofArtificial Sequence Peptide 12 Xaa His Trp Ser Tyr Xaa Leu Arg Xaa 1 513 9 PRT Artificial Sequence Description of Artificial Sequence Peptide13 Xaa His Trp Ser Xaa Xaa Xaa Arg Xaa 1 5 14 9 PRT Artificial SequenceDescription of Artificial Sequence Peptide 14 Xaa His Trp Ser Tyr XaaLeu Xaa Xaa 1 5 15 3 PRT Artificial Sequence Description of ArtificialSequence Peptide 15 Xaa Xaa Xaa

We claim:
 1. A method of producing a peptide (I) consisting of the aminoacid sequence of SEQ ID No: 6 or its salts, which method comprisesreacting a peptide (II) consisting of the amino acid sequence of SEQ IDNo: 4 or its salts, with a peptide (III) consisting of the amino acidsequence of SEQ ID No: 15 or its salts to produce a peptide (I′)consisting of the amino acid sequence of SEQ ID No: 5 or its salts,followed by subjecting the peptide (I′) to a de-protecting groupreaction using an acid to obtain the peptide (I), said acid being C₁₋₆alkanesulfonic acid and being used at a ratio of about 5 to about 25times (weight) of the peptide (I′), and followed by neutralizing theacid with a base at a temperature of about 15° C. to about −15° C. 2.The method according to claim 1, wherein (a) amino acid residue 2 of SEQID Nos: 4, 5 and 6 is His, (b) amino acid residue 5 of SEQ ID Nos: 4, 5and 6 is Tyr, (c) amino acid residue 6 of SEQ ID Nos: 4, 5 and 6 is Gly,D—Leu, D—Trp, D—Val which may be substituted with C₁₋₄ alkyl, D—Ser,D—Ala which may be substituted with C₁₋₄ alkoxy, with naphthyl or with2-methylindolyl, or D—His which may be substituted with C₇₋₁₀ aralkyl,(d) amino acid residue 7 of SEQ ID Nos: 5 and 6, and amino acid residueI of SEQ ID No: 15 is Leu, (e) amino acid residue 8 of SEQ ID No: 5, andamino acid residue 2 of SEQ ID No: 15 is Arg which is protected with agroup selected from the group consisting of a C₁₋₆ alkoxybenzenesulfonylgroup, a tri-C₁₋₆ alkylbenzenesulfonyl group and a nitro group, and (f)amino acid residue 9 of SEQ ID Nos: 5 and 6, and amino acid residue 3 ofSEQ ID No: 15 is group, Pro—NH—R″, wherein R″ represents a hydrogen atomor an alkyl group which may optionally be substituted with hydroxyl. 3.The method according to claim 1, wherein (a) amino acid residue 2 of SEQID Nos: 4, 5 and 6 is His, (b) amino acid residue 5 of SEQ ID Nos: 4, 5and 6 is Tyr, (c) amino acid residue 6 of SEQ ID Nos: 4, 5 and 6 isD—Leu, (d) amino acid residue 7 of SEQ ID Nos: 5 and 6, and amino acidresidue 1 of SEQ ID No: 15 is Leu, (e) amino acid residue 8 of SEQ IDNo: 5, and amino acid residue 2 of SEQ ID No: 15 is Arg which isprotected with a C₁₋₆ alkoxybenzenesulfonyl group, and (f) amino acidresidue 9 of SEQ ID Nos: 5 and 6, and amino acid residue 3 of SEQ ID No:15 is group, Pro—NH—R″, wherein R″ represents a C₁₋₃ alkyl group whichmay optionally be substituted with hydroxyl.
 4. The method according toclaim 1, wherein the reaction of the peptide (II) or its salts with thepeptide (III) or its salts is carried out at a temperature ranging fromabout 0° C. to about 40° C. for about 30 to about 60 hours.
 5. A methodof recovering and purifying a peptide (I) consisting of the amino acidsequence of SEQ ID No: 6 or its salts, which method comprises:subjecting a peptide (I′) consisting of the amino acid sequence of SEQID No: 5 or its salts to a de-protecting group reaction in the presenceof an acid to obtain peptide (I), said acid being C₁₋₆ alkanesulfonicacid, neutralizing the acid with a base, and purifying by columnchromatography peptide (I) to recover the peptide (I).
 6. A crystal of apeptide (II) consisting of the amono acid sequence of SEQ ID NO: 4, orits salt.
 7. A method of producing a crystal of a peptide (II)consisting of the amino acid sequence of SEQ ID NO: 4, or its salt,which method comprises cooling a solution containing peptide (II) toform a peptide (II) crystal.
 8. The method according to claim 7, furthercomprising adding seed crystals during cooling to facilitatecystalization.
 9. The method according to claim 7, wherein the solutionof the peptide (II) or its salt is subjected to aging.
 10. The methodaccording to claim 9, wherein the solution of the peptide (II) or itssalt with a concentration of about 0.01 to about 0.05 mole/liter issubjected to aging at a temperature ranging from about 10° C. to about70° C. for about 10 to about 70 hours.